While the pathogenesis of idiopathic Parkinson's Disease remains enigmatic, there is considerable evidence associating the cognition with heightened oxidative stress. Although the agent(s) responsible has not been elucidated, a plausible candidate is dopamine itself, which readily oxidizes to produce quinones and other free radicals, and is more toxic to cultured neurons than similar concentrations of MPTP. Toxic oxyradical dopamine metabolites may attack essential cytosolic proteins, providing a signal that renders them subject to ubiquitin-mediated proteolysis and degradation. Ubiquitin-positive Lewy bodies are the characteristic cellular hallmarks of Parkinson's Disease and it is thought by some that these inclusions result from an accumulation of abnormal proteins, prominently including neurofilament proteins as well as alpha-synuclein and cdk5. It is therefore possible that aberrant dopamine oxidative metabolism not only contributes to the selective degeneration of substantia nigra neurons but also to formation of Lewy bodies, as well as playing a well-established role in neuromelanin formation in lysosomal/endosomal compartments. In this proposal, we outline experiments designed to test how aberrations in vesicular and cytosolic dopamine pools initiate degeneration of substantia nigra neurons in postnatally-derived cultures. We will use four tools to alter substantia nigra intracellular dopamine. 1) Substantia nigra cultures derived from VMAT2 knockout mice, which are unable to sequester dopamine in synaptic vesicles. 2) Methamphetamine, which redistributes dopamine from synaptic vesicles to the cytosol, increasing the cytosolic pool and the transmitter available for release and eventually results in oxyradical-mediated synaptic degeneration. 3) L-DOPA, which is rapidly converted to cytosolic dopamine, resulting in an elevated vesicular dopamine pool and quantal size (the number of molecules released per synaptic vesicle exocytosis). We will also use neuronal cultures derived from alpha-synuclein knockout mice to determine if this protein is involved in biosynthesis of neuromelanin and ubiquitin inclusions. Remarkably, these approaches can be used to induce formation of intracellular neuromelanin and intracellular ubiquitinated inclusions, providing the first in vitro system for study of these features. These studies promise to provide information on neurodegeneration and the cell biology related to Parkinson's Disease in a preparation that provides the opportunity to study interventions in a living, malleable system.